Pretargeted PET Imaging with a TCO-Conjugated Anti-CD44v6 Chimeric mAb U36 and [89Zr]Zr-DFO-PEG5-Tz

The recent advances in the production of engineered antibodies have facilitated the development and application of tailored, target-specific antibodies. Positron emission tomography (PET) of these antibody-based drug candidates can help to better understand their in vivo behavior. In this study, we report an in vivo proof-of-concept pretargeted immuno-PET study where we compare a pretargeting vs targeted approach using a new 89Zr-labeled tetrazine as a bio-orthogonal ligand in an inverse electron demand Diels–Alder (IEDDA) in vivo click reaction. A CD44v6-selective chimeric monoclonal U36 was selected as the targeting antibody because it has potential in immuno-PET imaging of head-and-neck squamous cell carcinoma (HNSCC). Zirconium-89 (t1/2 = 78.41 h) was selected as the radionuclide of choice to be able to make a head-to-head comparison of the pretargeted and targeted approaches. [89Zr]Zr-DFO-PEG5-Tz ([89Zr]Zr-3) was synthesized and used in pretargeted PET imaging of HNSCC xenografts (VU-SCC-OE) at 24 and 48 h after administration of a trans-cyclooctene (TCO)-functionalized U36. The pretargeted approach resulted in lower absolute tumor uptake than the targeted approach (1.5 ± 0.2 vs 17.1 ± 3.0% ID/g at 72 h p.i. U36) but with comparable tumor-to-non-target tissue ratios and significantly lower absorbed doses. In conclusion, anti-CD44v6 monoclonal antibody U36 was successfully used for 89Zr-immuno-PET imaging of HNSCC xenograft tumors using both a targeted and pretargeted approach. The results not only support the utility of the pretargeted approach in immuno-PET imaging but also demonstrate the challenges in achieving optimal in vivo IEDDA reaction efficiencies in relation to antibody pharmacokinetics.


■ INTRODUCTION
Quantitative positron emission tomography (PET) imaging can be used in preclinical as well as clinical research and provides important information about the pharmacokinetics of monoclonal antibodies (mAbs) and derivatives thereof, particularly with respect to the kinetics of tumor accumulation and washout from nontarget tissues. 1 During the last decades, many antibodies have been developed for cancer diagnosis and treatment, and recent advances in the production of tailored antibodies for specific targets have provided several new radioimmunoconjugate candidates for immuno-PET imaging. 2−4 These second-generation radioimmunoconjugates can be grouped into different categories: (i) antibody−drug conjugates (ADCs), designed to release a drug when reaching its target; 5,6 (ii) multispecific mAbs, recognizing two or more targets; 7 (iii) glycoengineered mAbs, which are modified to enhance the antibody-dependent cytotoxicity; 8 and (iv) mAb fragments and nanobodies to tailor the radioimmunoconjugate pharmacokinetics. 9 The relatively slow pharmacokinetics of antibodies require that the radioactive half-life of the isotope must be compatible with the biological half-life of the mAb. In practice, this means that for immuno-PET imaging the antibodies are often labeled with isotopes with long, even multiday physical half-lives such as 89 Zr (78.41 h), 64 Cu (12.70 h), and 124 I (4.18 d), 10−12 which allows for the detection of the radiolabeled antibodies after accumulation at the tumor and clearance from the circulation. 13 It usually takes several days until nonbound antibodies are cleared from the circulation, and the optimal target-to-non-target (T:NT) values are obtained for imaging. 14,15 The administered radioactive dose can therefore be high. The levels of radiolabeled mAbs in blood can be reduced using special clearing agents; 16 however, this does not solve the problem of slow accumulation kinetics of mAbs in the tumor. Achieving high target-to-non-target values more rapidly would minimize the lag time needed between the radiotracer injection and the PET imaging, reducing exposure of the patient to radioactivity and the effective dose. Significant efforts have been dedicated to overcome these obstacles through the development of engineered antibody variants with faster pharmacokinetics and pretargeted approaches for radiolabeling the antibodies in vivo after their administration and peak accumulation to the target site. 12 Recently, in vivo click reactions based on the bioorthogonal inverse electron demand Diels−Alder ligation (IEDDA) between dienophile-functionalized antibodies and small-molecule radioligands based on tetrazine structures have obtained high interest. 17−22 Pretargeted immuno-PET imaging would bring significant advantages: reducing the radioactive exposure of the patients and allowing the use of the short halflive radionuclides for imaging purposes ( Figure 1). 12,23 The preclinical proof of concept of the two-step pretargeted immuno-PET imaging and radioimmunotherapy with IEDDA have been successfully achieved by several research groups. 17,24−27 Bio-orthogonal click reactions are specific and selective reactions that can take place under physiological conditions and rapidly react even at low concentrations in vivo. Fast reaction kinetics and selectivity have made them a favorable choice for effective in vivo radiolabeling methods for pretargeted imaging and therapy. 28 The IEDDA ligation between olefins or alkynes (e.g., trans-cyclooctene or TCO) and 1,2,4,5-tetrazines (e.g., tetrazine or Tz) is a selective, fast, high-yielding, biocompatible, and bio-orthogonal reaction, in which the reaction counterparts will undergo two concerted reactions to afford a coupling product under the formation of a pyridazine and dinitrogen ( Figure 1). Reaction between TCO and Tz holds one of the fastest reaction kinetics from all click chemistry methods, which makes them ideal functional groups for in vivo applications. Rate constants for the reaction between tetrazine and TCO can exceed 100,000 M −1 s −1 , orders of magnitude faster than either the Staudinger or strain-promoted azide−alkyne cycloaddition ligations. 29 Rossin et al. used the IEDDA for the first time for pretargeted SPECT imaging, and the first pretargeted PET study was reported by Weissleder and Lewis. 18,30 TCO isomerizes quickly to a less reactive cis- Pretargeting method based on an inverse electron demand Diels−Alder (IEDDA) ligation between trans-cyclooctene (TCO) and tetrazine. In the first step (a), a TCO-conjugated antibody is administered and allowed to reach the target, while unbound antibodies are slowly cleared from the circulation. In the second step (b), a radiolabeled tetrazine is administered and it reacts with the TCO-antibody. Unreacted tetrazine molecules are cleared fast from circulation. The radiolabeled antibody (c) is now visible compared to the nontarget tissue since most of the detected radioactivity signals originate from the tumor.
Bioconjugate Chemistry pubs.acs.org/bc Article cyclooctene (CCO) in vivo unless conjugated to a macromolecule; therefore, most of the published pretargeting studies are based on the IEDDA ligation between a TCO-conjugated antibody and a small-molecular tetrazine carrying the radiolabel.
In this study, a 89 Zr-labeled tetrazine ([ 89 Zr]Zr-DFO-PEG 5 -Tz, [ 89 Zr]Zr-3) was developed and utilized as a tool for investigation and comparison of targeted and pretargeted PET imaging of head-and-neck squamous cell carcinoma (VU-SCC-OE) xenografts using an anti-CD44v6 chimeric mAb (cmAb) U36. 31 U36 was chosen for the study because it has shown high and selective tumor uptake in head-and-neck squamous cell carcinoma (HNSCC) patients and it internalizes into cells only to a limited extent. 31 The splice variant v6 of the cell membrane glycoprotein CD44 (CD44v6) is expressed only in a few normal epithelial tissues (e.g., thyroid and prostate gland), 32 but it plays a significant role in solid tumor growth and metastasis development. For the HNSCC, >96% of tumors show CD44v6 expression by at least 50% of the cells. 33 In addition to squamous cell carcinomas, CD44v6 is overexpressed in adenocarcinomas and ovarian cancer and in hematological tumors. 34−36 Expression of CD44v6 in tumors has been imaged by several research groups using U36 or its variants after radiolabeling it with different long-living radionuclides. 37−40 In this study, U36 was conjugated with trans-cyclooctene and the conjugation ratio was optimized with biodistribution studies. TCO−U36 was radiolabeled in vitro and in vivo using [ 89 Zr]Zr-3, and the uptake levels in VU-SCC-OE tumors were quantified with PET-CT/MRI and ex vivo biodistribution studies.
Immunoreactivity of [ 89 Zr]Zr-3−TCO−U36 with CD44v6. Immunoreactivity of [ 89 Zr]Zr-3−TCO−U36 was determined using CD44v6-coated beads using TCO-conjugated U36 with the highest TCO-to-U36 ratio (27:1). Despite the high TCO-to-U36 ratio, immunoreactivity was well preserved with a 91.6 ± 1.3% immunoreactivity corrected for nonspecific binding at a CD44v6 bead concentration of 1.6 × 10 6 /mL (n = 3) ( Figure S1).   The results revealed that the pharmacokinetics of the antibody were significantly altered due to the excessive TCO conjugation ( Figure 3 and Table S2). Liver uptake for the in vitro-labeled [ 89 Zr]Zr-3−TCO−U36 was high (14.1 ± 2.9% ID/g at 72 h p.i.), and tumor uptake was lower (6.1 ± 1.1% ID/g at 72 h p.i.) compared to the results previously reported by Vugts et al. using the same mAb dose (0.1 mg, azide conjugation ratio 4:1; liver: 3.9 ± 0.4% ID/g and tumor: 23.1 ± 3.4% ID/g at 72 h p.i.). 41 However, the initial results confirmed successful in vivo IEDDA reaction with the highest tumor uptake of 3.3 ± 0.5% ID/g at 72 h when the tracer [ 89 Zr]Zr-3 was injected at 24 h p.i. TCO−U36 and 1.5 ± 0.6% ID/g when injected at 48 h p.i. TCO−U36. The results indicate that the maximum 50% of TCO−U36 reaching the tumor at 72 h was radiolabeled in vivo since tumor accumulation of the in vitro-labeled [ 89 Zr]Zr-3−TCO−U36 was 6.11 ± 1.12% ID/g at 72 h. It was therefore evident that further optimization of the TCO-to-mAb ratio was needed for minimizing the effect of the TCO conjugation on the pharmacokinetics of the antibody.
Ex Vivo Biodistribution of [ 89 Zr]Zr-3−TCO−U36 with Different TCO Conjugation Ratios in Non-Tumor-Bearing Animals. Biodistribution of the [ 89 Zr]Zr-3-labeled U36 was investigated with varying TCO-to-U36 ratios and compared to the biodistribution of 125 I-labeled U36 without any TCO groups attached. Ex vivo biodistribution at 72 h p.i. showed clearly how the TCO-to-U36 ratio affected the liver uptake of the antibody and how the blood radioactivity levels increased with decreasing antibody accumulation in the liver (Figure 4). With a TCO-to-U36 ratio of 10:1, the lowest liver uptake and the highest radioactivity in the circulation were obtained.  A clear correlation was observed between the increased liver uptake and decreased blood concentrations when more TCO moieties were conjugated to U36 (Pearson correlation coefficient R for liver = 99.3 and for blood = −68.6) ( Figure  5). The effect of small-molecule conjugation on the U36 antibody pharmacokinetics was surprisingly high compared to the finding of the reported study by Vugts et al. with a phenolic PEG 5 -triazide-conjugated U36, where the influence of the azide conjugation to liver accumulation and to clearance from blood was less prominent even with a ratio of 15 azides on 1 U36. 41 Therefore, we decided to repeat the pretargeted PET study with even a lower TCO-to-U36 ratio than 10:1 with the goal of further decreasing the observed liver uptake.
In Vivo Evaluation of TCO−U36 with a 6:1 TCO-to-U36 Ratio in VU-SCC-OE Xenografts. Using the same experimental setup as used in the initial biological evaluation, the ex vivo biodistribution data showed improved pharmacokinetics of [ 89 Zr]Zr-3−TCO−U36 with a typical, high tumor accumulation of 17.1 ± 3.0% ID/g and a low liver uptake of 5.5 ± 1.1% ID/g at 72 h p.i. ( Figure 6A and Table S2). However, tumor uptake in the pretargeted approach was lower: 1.6 ± 0.3% ID/g when [ 89 Zr]Zr-3 was injected at 24 h p.i. of U36 and 1.5 ± 0.2% ID/g when injected at 48 h p.i. of U36  Although the tumor uptake values were significantly lower with the pretargeted approach, the same tumor-to-background ratios were achieved when compared to the in vitro-labeled U36 (Table 1). For the in vitro-labeled U36, the tumor-tomuscle ratio was 25.67 ± 6.30, and for the in vivo pretargeting, the ratio was 23.49 ± 6.22 when the tracer was injected 24 h p.i. of the TCO−U36. The tumor uptake was slightly lower when the tracer was injected 48 h p.i. of TCO−U36, resulting in a lower tumor-to-muscle ratio of 15.56 ± 6.57.
Despite the lower activity concentration in the pretargeted tumors, the tumors were clearly visible by PET-computed tomography (PET/CT) due to the low background activity (Figure 7). Tumor activities were quantified by delineating region of interests around the tumors and by calculating standardized uptake values (SUVs) for all groups at 1, 24, 48, and 71 h after the U36 injection ( Figure 8). The tumor volumes varied from 31 to 793 mm 3 , and the heterogeneous structure of the tumors caused some additional challenge for the image analysis and calculation of the SUVs. Due to the structural heterogeneity (necrotic core poorly perfused), the activity concentrations varied significantly between the tumors, resulting in high variation of the SUVs between tumors from the same group. In general, small tumors (<100 mm 3 ) had clearly higher activity concentration compared to the larger ones (Table S1).
Volume  (Table 2). Especially, for the few important organs, the absorbed dose difference was significant between the pretargeted U36 and the in vitrolabeled U36 groups, for example, in the heart (0.086 and 0.072 vs 0.471 for 24 h pretargeted, 48 h pretargeted, and in vitrolabeled groups, respectively), liver (0.123 and 0.082 vs 0.970), and spleen (0.057 and 0.054 vs 0.395). There was also a considerable difference between the two approaches when considering the absorbed dose to the bone. Dose values for red marrow and osteogenic cells were approximately 5 times lower with the pretargeted approach. The dose estimations for the in vitro-labeled U36 were in line with the results that were reported by Borjesson and co-workers with 89 Zr-labeled U36 in humans. 42 Although the values from the human study were higher (liver 1.

■ DISCUSSION
In this study, we investigated the pretargeted PET imaging of VU-SCC-OE xenografts utilizing the IEDDA reaction between a zirconium-89-labeled tetrazine ([ 89 Zr]Zr-3) and a TCOfunctionalized anti-CD44v6 antibody U36. The relatively long half-life (t 1/2 = 78.41 h) of zirconium-89 enabled the direct comparison of the tumor targeting in vivo with in vitro-labeled U36 and after pretargeting of TCO-modified U36. U36 was chosen for the study because it has shown high and selective tumor uptake in head-and-neck squamous cell carcinoma patients and it internalizes into cells only to a limited extent. 31 Both properties are favorable for successful pretargeting. In   with a reasonable tumor uptake of 3.3 ± 0.5% ID/g when a high TCO-to-U36 ratio (27:1) was used in the antibody conjugation. However, the higher TCO-to-U36 ratio had its drawbacks as it significantly increased the liver accumulation of the U36 due to the altered pharmacokinetics of the functionalized antibody and increased the clearance from the blood. Decreasing the TCO-to-U36 ratio from 27:1 to 6:1 successfully reduced the unfavorable liver uptake by two-thirds but also resulted in lower tumor accumulation (1.5 ± 0.2% ID/ g at 72 h). This may be explained by the lower IEDDA reaction efficiency at the lower TCO-to-U36 ratio. In pretargeted PET imaging applications, fast reaction kinetics at low concentrations are required for efficient in vivo labeling. 43 The IEDDA reaction is characterized by the second-order reaction kinetics with dependence on concentration of the reactants, in our case, the TCO concentration at the target site. Decreasing the TCO-to-U36 ratio from 27:1 to 6:1 increased the tumor accumulation of the in vitroradiolabeled U36 from 6.11 ± 1.12 to 17.1 ± 3.0% ID/g but resulted in a lower tumor accumulation in the pretargeted approach. Obviously, the 2.8 times higher antibody concentration in the tumor was not enough to compensate for the lower TCO-to-U36 ratio in vivo, resulting in lower TCO concentration in the tumor and consequently lower in vivo IEDDA reaction efficiency in the pretargeted approach. In addition, the higher TCO-mAb levels in blood were most probably contributed by consuming the [ 89 Zr]Zr-3 before it reached the tumor site. Another explanation for the lower IEDDA reactivity could be the in vivo deactivation of TCO. Deactivation of TCO by isomerization in the presence of high thiol concentrations has been reported, leading to decreased in vivo reactivity and consequently lower tumor activities. Robillard et al. showed that in fresh mouse serum at 37°C the trans-isomer converts into cis-cyclooctene with a half-life of 3.26 h. By attaching the TCO through a short linker, as done in this study, the deactivation half-life of TCO in circulation in mice was increased to 4 days. 44 Indeed, we did not observe any statistically significant decrease in TCO reactivity between the groups that received [ 89 Zr]Zr-3 at 24 and 48 h p.i. when the lower TCO-to-U36 ratio was used. With the higher 27:1 TCOto-U36 ratio, lower tumor activity was observed at the later time point, but this can be rather attributed to the altered pharmacokinetics of TCO−U36 at a high degree of conjugation than the in vivo isomerization of the TCO in this case.
In vivo IEDDA reaction yields can be improved by increasing the TCO concentration at the target site. However, as demonstrated by our results and reported previously by others, increasing the TCO-to-mAb conjugation ratio has its limitations since the pharmacokinetics of the antibody can be altered when too high conjugation ratios are used. 45,46 When compared to the previous study with triazide-conjugated U36, 41 the change in pharmacokinetics in the current study was mainly evidenced by the decreased tumor and blood radioactivity levels and increased liver uptake upon increasing the TCO-to-U36 ratio. This is most likely because of the increased lipophilicity of the antibody due to the conjugation.
Stability of the radiolabeled [ 89 Zr]Zr-3 in formulation solution, diluted in 10% EtOH in saline + 0.1% Tween, 20 mM gentisic acid, pH = 5.2, was measured after 4, 24, and 48 h storage at°C, and stability was measured with iTLC-SG and HPLC (Alltima C18). . The radiolabeling yield was 85 ± 4%, and the radiochemical purity was >99%. Immunoreactivity of TCO−U36. Immunoreactivity of the TCO-conjugated U36 (27.2 TCO-to-U36) was analyzed with CD44v6-coated superparamagnetic immuno-beads. The binding experiment was done in triplicate with five bead concentrations (2.5 × 10 7 to 1.6 × 10 6 /mL) in a 1% bovine serum albumin (BSA) in PBS solution and in one control for nonspecific binding with a bead concentration of 1.6 × 10 6 /mL, essentially as described by Lindmo et al. 47  were imaged with PET-CT/MRI at 1 (dynamic scan), 24, 48, and 71 h after U36 injection, group 2 mice were imaged 1 (dynamic scan), 24, and 47 h, and group 3 were imaged 1 (dynamic scan) and 23 h after the injection of the tracer. All mice were sacrificed at 72 h p.i. of the U36 injection, and the collected organs (urine, blood, gall bladder, pancreas, spleen, kidney, liver, heart, lung, stomach, small intestine, large intestine + cecum, feces (1−2 pellets from the rectum), bladder, skeletal muscle, bone (tibia), bone (skull), brain, skin,  ) with TCO-to-U36 ratios between 9:1 and 15:1 were injected i.v. (200 μL, saline). All mice were sacrificed at 72 h p.i., and the harvested organs (same as above) were weighed and the amount of radioactivity in each tissue was measured by a γ-counter. Radioactivity uptake was calculated as the percentage of the injected dose per gram of tissue (% ID/g).
Biodistribution Study of In Vitro-Labeled TCO−U36 and In Vivo Click Reaction (6:1 TCO-to-U36). Experiments were done in nude female mice (HSD:athymic nude Foxn1 nu , 15− 30 g; Envigo, Horst, the Netherlands), aged 8−10 weeks at the time of the experiment, bearing subcutaneously implanted VU-SCC-OE xenografts (tumor volumes varied from 31 to 793 mm 3 ). Mice were randomized to three groups as described above. At day 1, group 1 mice were injected (i.v.) with in vitro- Group 1 mice were imaged with PET-CT at 1 (dynamic scan), 24, 48, and 71 h after cmAb injection, group 2 mice were imaged 1 (dynamic scan), 24, and 47 h, and group 3 were imaged 1 (dynamic scan) and 23 h after injection of the tracer. All mice were sacrificed at 72 h p.i. of U36, and the collected organs (same as above) were weighted and the amount of radioactivity in each tissue was measured by a γ-counter. Radioactivity uptake was calculated as the percentage of the injected dose per gram of tissue (% ID/g). Quantitative PET image analysis was performed by defining regions of interest (ROIs) around the tumor with CT as the anatomical reference. Radioactivity concentration was expressed as an SUV, calculated using the average radioactivity concentration of the ROI normalized with the injected radioactivity dose and animal weight.
Organ Dosimetry. The activity for each organ that was visible in PET/CT scans (heart, liver, lungs, spleen, kidneys, small intestine, large intestine, bladder, bone, and muscle) was determined using the mean activity concentration in VOIs with Vinci64 v 5.06 software. VOIs were independently drawn on all PET/CT scans for each mouse. The total activity in each organ was then calculated from the activity concentration and the Olinda 25 g mice model organ weight. Organ time−activity curves were created by collating the total activity from all mice fitted by exponential functions. Analytical integration of the fit provided the organ residence times, and this data was used as an input in OLINDA/EXM 2.1. This software was used for the calculation of organ-absorbed doses and the effective dose. Human dosimetry estimates were obtained from the residence times using OLINDA/EXM version 2.1 software with the adult model.
Statistics. The statistical difference was evaluated by Student's t-test, where the significant probabilities were set at *p < 0.05, **p < 0.01, and ***p < 0.001.